Technical Resources

Introduction to AAV

Adeno-associated virus (AAV) is a small virus which infects humans and some
other primate species. AAV belongs to the genus Dependovirus, which in turn
belongs to the family Parvoviridae. The virus is a small (20 nm)
replication-defective, nonenveloped virus. AAV is not currently known to
cause disease and consequently the virus causes a very mild immune response.
Wild type AAV
can infect both dividing and non-dividing cells and may incorporate its genome
into that of the host cell. These features make AAV a very attractive candidate
for creating viral vectors for gene therapy, and for the creation of isogenic
human disease models.

AAV Structure:- AAV genome, transcriptome and proteomeThe AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either
positive- or negative-sensed, which is about 4.7 kilobase long. The genome
comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and
two open reading frames (ORFs): rep and cap. The former is composed of four
overlapping genes encoding Rep proteins required for the AAV life cycle, and the
latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2
and VP3, which interact together to form a capsid of an icosahedral symmetry
(Figure 1).

- ITR sequencesThe Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were
named so because of their symmetry, which was shown to be required for efficient
multiplication of the AAV genome. Another property of these sequences is
their ability to form a hairpin, which contributes to so-called self-priming
that allows primase-independent synthesis of the second DNA strand. The ITRs
were also shown to be required for both integration of the AAV DNA into the host
cell genome (19th chromosome in humans) and rescue from it as well as
for efficient encapsidation of the AAV DNA combined with generation of a fully
assembled, deoxyribonuclease-resistant AAV particles (Figure 2).
With regard to gene therapy, ITRs seem to be the only sequences required in cis
next to the therapeutic gene: structural (cap) and packaging (rep) genes can be
delivered in trans. With this assumption many methods were established for
efficient production of recombinant AAV (rAAV) vectors containing a reporter or
therapeutic gene. However, it was also published that the ITRs are not the only
elements required in cis for the effective replication and encapsidation. A few
research groups have identified a sequence designated cis-acting Rep-dependent
element (CARE) inside the coding sequence of the rep gene. CARE was shown to
augment the replication and encapsidation when present in cis.Figure 2. Secondary structure of the AAV2
ITR. The AAV2 ITR serves as origin of replication and is composed of two arm
palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A'). The ITR
can acquire two configurations (flip and flop). The flip (depicted) and flop
configurations have the B-B' and the C-C' palindrome closest to the 3' end,
respectively. The D sequence is present only once at each end of the genome thus
remaining single-stranded. The boxed motif corresponds to the Rep-binding
element (RBE) where the AAV Rep78 and Rep68 proteins bind. The RBE
consists of a tetranucleotide repeat with the consensus sequence 5'-GNGC-3'. The
ATP-dependent DNA helicase activities of Rep78 and Rep68 remodel the A-A' region
generating a stem-loop that locates at the summit the terminal resolution site (trs)
in a single-stranded form. In this configuration, the strand- and site-specific
endonuclease catalytic domain of Rep78 and Rep68 introduces a nick at the trs.
The shaded nucleotides at the apex of the T-shaped structure correspond to an
additional RBE (RBE') that stabilizes the association between the two largest
Rep proteins and the ITR.

- Rep genes and Rep proteinsOn the "left side" of the genome there are two promoters called p5 and p19, from
which two overlapping messenger ribonucleic acids (mRNAs) of different length
can be produced. Each of these contains an intron which can be either spliced
out or not. Given these possibilities, four various mRNAs, and consequently four
various Rep proteins with overlapping sequence can be synthesized. Their names
depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78
and 68 can specifically bind the hairpin formed by the ITR in the self-priming
act and cleave at a specific region, designated terminal resolution site, within
the hairpin. They were also shown to be necessary for the AAVS1-specific
integration of the AAV genome. All four Rep proteins were shown to bind ATP and
to possess helicase activity. It was also shown that they upregulate the
transcription from the p40 promoter (mentioned below), but downregulate both p5
and p19 promoters (Figure 3).

- Cap genes and VP proteinsThe right side of a positive-sensed AAV genome encodes overlapping sequences
of three capsid proteins, VP1, VP2 and VP3, which start from one promoter,
designated p40. The molecular weights of these proteins are 87, 72 and 62
kiloDaltons, respectively. All three of them are translated from one mRNA. After
this mRNA is synthesized, it can be spliced in two different manners: either a
longer or shorter intron can be excised resulting in the formation of two pools
of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the
presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA
represents the so-called "major splice". In this form the first AUG codon, from
which the synthesis of VP1 protein starts, is cut out, resulting in a reduced
overall level of VP1 protein synthesis. The first AUG codon that remains in the
major splice is the initiation codon for VP3 protein. However, upstream of that
codon in the same open reading frame lies an ACG sequence (encoding threonine)
which is surrounded by an optimal Kozak context. This contributes to a low level
of synthesis of VP2 protein, which is actually VP3 protein with additional N
terminal residues, as is VP1.
Since the bigger intron is preferred to be spliced out, and since in the major
splice the ACG codon is a much weaker translation initiation signal, the ratio
at which the AAV structural proteins are synthesized in vivo is about 1:1:20,
which is the same as in the mature virus particle. The unique fragment at the N
terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2)
activity, which is probably required for the releasing of AAV particles from
late endosomes. It was reported that VP2 and VP3 are crucial for
correct virion assembly. More recently, however, Warrington et al. showed VP2 to
be unnecessary for the complete virus particle formation and an efficient
infectivity, and also presented that VP2 can tolerate large insertions in its N
terminus, while VP1 can not, probably because of the PLA2 domain presence.
The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3,
that are arranged in a icosahedral symmetry in a ratio of 1:1:10, with an
estimated size of 3,900 KiloDaltons (Figure 3). Figure 3.
A cartoon showing transcription and translation of Rep and Cap genes to Rep and
Cap proteins.

AAV Serotypes, Receptors and Native Tropism:So far there are about total 12 AAV serotypes described
and reported. All of
the known serotypes can infect cells from multiple diverse tissue types. Tissue
specificity is determined by the capsid serotype and pseudotyping of AAV vectors
to alter their tropism range will likely be important to their use in therapy.

- Serotype 2Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents
natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells
and hepatocytes.
Three cell receptors have been described for AAV2: heparan sulfate proteoglycan
(HSPG), aVβ5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The
first functions as a primary receptor, while the latter two have a co-receptor
activity and enable AAV to enter the cell by receptor-mediated endocytosis. HSPG functions as
the primary receptor, though its abundance in the extracellular matrix can
scavenge AAV particles and impair the infection efficiency (Figure 4).

- Other SerotypesAlthough AAV2 is the most popular and most
studies serotype in various AAV-based research, it
has been shown that other serotypes can be more effective as gene delivery
vectors. For instance AAV6 appears much better in infecting airway epithelial
cells, AAV7 presents very high transduction rate of murine skeletal muscle cells
(similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes and AAV1
and 5 were shown to be very efficient in gene delivery to vascular endothelial
cells. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than
AAV2.
Serotypes can differ with the respect to the receptors they are bound to. For
example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of
different form for each of these serotypes), and AAV5 was shown to enter cells
via the platelet-derived growth factor receptor (Table 1).

Table 1. AAV serotypes and their tropisms.

Serotype

CNS/Retina

Heart

Lung

Liver

Skeletal Muscle

AAV1

X

X

X

X

AAV2

X

X

X

AAV3

X

X

X

AAV4

X

X

AAV5

X

X

AAV6

X

X

X

X

AAV7

X

X

X

AAV8

X

X

X

AAV9

X

X

X

X

X

AAV10

X

X

AAV immunologyAAV is of particular interest to gene
therapists due to its apparent limited capacity to induce immune responses in
humans, a factor which should positively influence vector transduction
efficiency while reducing the risk of any immune-associated pathology. The innate immune response to the AAV vectors has been characterised in
animal models. Intravenous administration in mice causes transient production of
pro-inflammatory cytokines and some infiltration of neutrophils and other
leukocytes into the liver.

- Cell-mediatedThe cell-mediated response to the virus and to vectors is poorly
characterised and has been largely ignored in the literature as recently as
2005. Clinical trials using an AAV2-based vector to treat haemophilia B seem to
indicate that targeted destruction of transduced cells may be occurring.
Combined with data that shows that CD8+ T-cells can recognise elements of the
AAV capsid in vitro, it appears that there may be a cytotoxic T lymphocyte
response to AAV vectors. Cytotoxic responses would imply the involvement of CD4+
T helper cells in the response to AAV and in vitro data from human studies
suggests that the virus may indeed induce such responses including both Th1 and
Th2 memory responses. A number of candidate T cell stimulating epitopes have
been identified within the AAV capsid protein VP1, which may be attractive
targets for modification of the capsid if the virus is to be used as a vector
for gene therapy.

How Is The Recombinant AAV (rAAV) Packaged?
Unlike wild type AAV, the recombinant AAV (rAAV) fails to integrate its genome
to host cells. With the newly developed rAAV packaging technology, rAAV
packaging nowadays does no reply on the presence of helper virus such as
adenovirus or Herpse. Instead by suing a helper-free system, rAAV can be
packaged easily by co-transfecting rAAV cis plasmid with Rep/Cap plasmid and
helper plasmid to a packaging cell. The helper plasmid will be simulating
helper virus by offering adenoviral E1, E2, E4 and VA RNAs during rAAV
packaging. rAAV is then produced in the packaging cell and can be
harvested 2 to 3 days after co-transfection via lysating packaging cells by
3xfreeze/thaw cycle. The rAAVs released into supernatant is then purified via
ultracentrifugation (Figure 5). Figure 5.
A cartoon showing how rAAV is packaged with a helper free system.

AAV Life Cycle:Wild type AAV undergoes productive infection in
the presence of adenovirus co-infection. This is characterized by genome
replication, viral gene expression, and virion production. In the absence of
adenovirus, AAV can establish latency by integrating into chromosome 19 (AAVS1).
The latent AAV genome can be rescued and replicated upon superinfection by
adenovirus. Both stages of AAV's life cycle are regulated by complex
interactions between the AAV genome and AAV, adenoviral, and host proteins
(Figure 6).Figure 6.
A cartoon showing AAV life cycle.

Clinical Trials with rAAV Vectors:
To date, AAV vectors have been used for first- and second-phase clinical trials
for treatment of cystic fibrosis and first-phase trials for hemophilia.
Promising results have been obtained from phase I trials for Parkinson's
disease, showing good tolerance of an AAV2 vector in the central nervous system.
Other trials have begun, concerning AAV safety for treatment of Canavan disease,
muscular dystrophy and late infantile neuronal ceroid lipofuscinosis (table 2).

Table 2. Selection of Clinical Trials using AAV-Based Vectors

Indication

Gene

Route of Administration

Phase

Subject Number

Status

Cystic fibrosis

CFTR

Lung, via aerosol

I

12

Complete

CFTR

Lung, via aerosol

II

38

Complete

CFTR

Lung, via aerosol

II

100

Complete

Hemophilia B

FIX

Intramuscular

I

9

Complete

FIX

Hepatic artery

I

6

Ended

Arthritis

TNFR:Fc

Intraarticular

I

1

Ongoing

Hereditary emphysema

AAT

Intramuscular

I

12

Ongoing

Muscular dystrophy

Sarcoglycan

Intramuscular

I

10

Ongoing

Parkinson's

GAD65, GAD67

Intracranial

I

12

Complete

Canavan's

AAC

Intracranial

I

21

Ongoing

Batten's

CLN2

Intracranial

I

10

Ongoing

Alzheimer's

NGF

Intracranial

I

6

Ongoing

Trials for the treatment of prostate cancer
have reached phase III, however these ex vivo studies do not involve direct
administration of AAV to patients.